Human tumors are usually not spontaneously eliminated by the immune system and therapeutic vaccination of cancer patients with defined antigens is followed by tumor regressions only in a small minority of the patients. The poor vaccination effectiveness could be explained by an immunosuppressive tumor microenvironment. Because T cells that infiltrate tumor metastases have an impaired ability to lyse target cells or to secrete cytokine, many researchers are trying to decipher the underlying immunosuppressive mechanisms. We will review these here, in particular those considered as potential therapeutic targets. A special attention will be given to galectins, a family of carbohydrate binding proteins. These lectins have often been implicated in inflammation and cancer and may be useful targets for the development of new anti-cancer therapies. Moreover, some of these expanded tumor-infiltrating lymphocyte (TIL) populations were re-injected in patients together with IL-2 and produced objective tumor responses [in vitro [Before the molecular identification of human tumor antigens, the existence of a spontaneous anti-tumor T cell response in melanoma patients was strongly suggested by the fact that CD8 cytolytic T cells (CTL), isolated from tumors and expanded for several weeks esponses . More rein vitro -5. FrequThe molecular proof of the existence of human tumor-specific antigens came with the cloning of the first gene encoding a human melanoma antigen recognized by CTL: MAGE-1 . The stiex vivo in the blood, even in those patients who showed tumor regressions, sensitive and quantitative approaches were set up allowing the detection of frequencies of CTL precursors specific for a MAGE-3 peptide presented by HLA-A1 (anti-MAGE-3.A1) as low as one precursor per million CD8 blood T cells [Most of these vaccines induced anti-vaccine T cells. The proportions of patients with detectable anti-vaccine T cells in their blood were highly variable. At first, no correlation was evidenced between immune T cell response to the vaccine and objective clinical response or tumor regressions -21. Beca T cells ,23. Notw T cells ,18,23: AIn addition to T cell frequencies in the blood, other parameters can be informative to evaluate the response to the vaccine. In patients vaccinated with mature dendritic cells, pulsed with gp100 and tyrosinase peptides, delayed-type hypersensitivity (DTH) was observed in all the patients after a subcutaneous challenge. Anti-gp-100 or anti-tyrosinase CTL were derived only from DTH sites of those patients who experienced tumor regression . Taken tNevertheless therapeutic vaccination of metastatic melanoma patients with these antigens is followed by tumor regressions of clinical significance in less than 10% of the patients. Even considering the absence of toxicity, the current vaccines are not effective enough to become generally applicable, as reviewed by different authors ,19,25,26In this review, we will focus on the T lymphocytes that infiltrate tumors. Can we consider T cell infiltration as a clinical prognostic factor? Are these TIL functional or anergic? What are the mechanisms orchestrated by the tumor that would result in T cell dysfunction? We do believe that the tumor micro-environment inhibits by the same mechanisms both the spontaneously generated anti-tumor T cells and the anti-vaccine T cells. Our hope is to better understand the immune suppression mechanisms orchestrated by the tumor so as to be able to improve the clinical efficacy of cancer vaccines.2.2.1.The correlation between patient survival and T cell-infiltrate in primary melanoma has been examined in about 20 papers published since the late seventies -47. In Tcompletely surrounding the part of the melanoma that invaded the normal tissues” [TIL were present throughout the substance of the vertical growth phase or present and infiltrating across the entire base of the vertical growth phase”. Since then, the general view was that a brisk T cell infiltration was a good clinical prognostic factor in melanoma.In the first study, Larsen and Grude reported in 1978 a better overall survival of the patients with an intense lymphocytic infiltration “tissues” . Ten yeatissues” . Clark aIn the nineties, the systematic biopsy of the sentinel lymph node (SLN) was introduced, which is the hypothetical first lymph node or group of nodes reached by metastasizing cancer cells from a primary melanoma . AbsenceIn melanoma metastases, higher numbers of TIL have been associated with a better survival ,36,46. C2.2.Beyond melanoma, there is growing evidence that dense TIL infiltrate is associated with a longer survival .In various histological types of tumors, the presence of TIL is unanimously considered as a good clinical prognostic factor: colon adenocarcinoma -59, ovarColon adenocarcinomas without any sign of metastasis had more T cell infiltrates than tumors with pathological signs of early metastatic invasion such as vascular emboli, lymphatic invasion or perineural invasion .i.e., injections of IL-2 and IFN-α, the fact that tumor infiltration by T cells is of bad prognosis seems contra-intuitive, but considering that TIL numbers usually increase with the tumor grade, the simplest explanation for this correlation is that the grade of the tumors were not taken into account. However, the positive impact of immune cells on tumor growth cannot be excluded.Presence of TIL is not always of good prognosis, e.g., in squamous cell carcinoma  and renaNoteworthy, TIL populations contain not only CD8 T cells able to kill tumor cells and CD4 T helper cells that secrete cytokines supporting proliferation of other T cells, but also regulatory T cells (Treg) able to inhibit function and proliferation of other T cells, and T cells that secrete, e.g., chemokines attracting macrophages and vasoendothelial growth factor (VEGF) promoting neoangiogenesis. In other words, TIL could also indirectly promote tumor progression .3.It is tempting to think that the survival advantage conferred by the presence of TIL is due to the presence of tumor-antigen specific T cells in the tumor. However, T cell trafficking to the tumor site is not antigen-specific, as it is due to the recruitment of blood circulating T cells that express the appropriate adhesion molecules and chemokine receptors. What are therefore the evidences for anti-tumor T cell accumulation in the tumor mass?3.1.T cell receptor (TCR) genomic loci undergo somatic recombination, plus the addition and removal of bases at recombination junctions, in order to generate the repertoire of structurally diverse TCR necessary for antigen recognition. The different TCR-β subunit chains, which have been estimated at 1 million per individual , can be Evidence for enrichment in one TCR transcript was followed for a few patients by molecular identification of the antigen recognized by the corresponding CTL, which was isolated either from the tumor or from the blood. This led to the identification of four MAGE-C2 peptides, one MAGE-6 peptide, one gp100 peptide and one peptide encoded by a mutated gene encoding myosin class I -112.in situ is the observation of tertiary lymphoid structures in some tumors. Tertiary lymphoid structures are lymph node-like structures inside the tumor mass composed of dendritic cells, T cells and B cells organized in germinal centers that could be sites of antigen presentation [et al. [Clearly in favor of TIL proliferation entation ,114. In  [et al. .3.2.in vitro [Study of the TCR diversity indicates accumulation of TCR clonotypes, suggesting the presence of anti-tumor T cells, but often without indication about the antigens recognized by these T cells. Hence the use of HLA-peptide multimers was introduced to identify some of the antigens targeted by TIL. We have only considered experiments with TIL that were not expanded before analysis. Using HLA-A2 multimers, which were folded with four peptides derived from melanoma differentiation proteins, the cumulative frequency of multimer-positive TIL ranged from 0 to >2% of the TIL . Interestingly, these frequencies of anti-tumor T cells were close to the frequencies observed for anti-hepatitis CD8 T cells detected in liver biopsies of patients chronically infected with hepatitis C virus. Using a pool of HLA-A2 multimers, which were folded with four peptides derived from non-structural proteins of hepatitis C, frequencies of multimer-positive T cells ranged from 0 to >2% of the TIL  . In othein vitro -118. In 3.3.The frequencies of anti-vaccine T cells infiltrating metastases of a melanoma patient, who was vaccinated with MAGE-3.A1 and experienced tumor regression, were analyzed in details. These frequencies were compared with frequencies of TIL directed against tumor antigens other than the vaccine antigens (referred to as “anti-tumor TIL”). No anti-vaccine T cells were detected before vaccination in the blood or in the tumor. After vaccination, the frequency of anti-vaccine T cells was 1/67,000 of CD8 T cells in an invaded lymph node, 6-fold higher than in the blood . After vTaken together, these results suggest that the anti-vaccine CTL are clearly not the main effectors that kill the bulk of the tumor cells, but that their interaction with the tumor generates conditions enabling at least part of the numerous anti-tumor CTL to participate to the destruction of the tumor cells. Naive T cells appear to be stimulated in the course of this process as new anti-tumor clonotypes arise after vaccination. Indeed, one CTL clonotype, specific for a peptide encoded by a mutated caseinolytic protease, represented up to 7% of the TIL in metastases, whereas the corresponding TCR clonotype was not detected before vaccination ,119,120.336-344.A2 T cell clonotype was detected in the blood and in the tumor before and after vaccination, but another anti-MAGE-C2336-344.A2, which recognized the same peptide/HLA was found at 1/11,000 in the blood only after vaccination and represented up to 9% of the CD8 TIL [An example of clonotype spreading—a diversification of the T cell clonotypes recognizing the same antigen—was also observed. An anti-MAGE-C2 CD8 TIL ,120. Clo CD8 TIL . This cl4.4.1.Expression of activation markers CD137 or CD25 on TIL was used as markers of TIL that have recently encountered their antigen. Expression of these markers in the peritumoral region was associated with a better survival ,122. ConTo detect TIL proliferating at the tumor site, the presence of nuclear protein Ki-67 was used as a marker of cell division . Higher 4.2.i.e., which form immunologic synapses, were in apoptosis whereas only 25% of the tumor cells that were not in contact with TIL displayed signs of apoptosis [The TUNEL method labels fragmented DNA, which is a characteristic of apoptotic cells. In seminoma, 2% of tumor cells were considered apoptotic by this method and half of them were in contact with at least one CD3 cell -126. Co-poptosis .4.3.+ CD8 TIL is a favorable prognosis factor [+ cells among the CD8 TIL can reach 30% [+ cells among the CD8 T cells in the liver of patients with hepatitis B, but it can reach 40% in hepatitis B patients that have an autoimmune disease characterized by T cell hyperreactivity [The consensus that seems to emerge from studies on colon adenocarcinoma, hepatocellular carcinoma, melanoma and lymphoma, is that the presence of granzyme Bs factor ,58,70,77each 30% . This fractivity -130.Upon activation, CTL express CD107a at their surface for a few hours. This lysosomal-associated membrane protein (LAMP-1 or CD107a) has been described as a marker of degranulation of CD8 T cell upon stimulation. This marker was found at the surface of 1% to 9% of the TIL in colon adenocarcinoma metastases and seminoma -133. Con4.4.The optimal cytokine pattern for an efficient anti-tumor immune response may differ in various tumor types. Cytokines have not been examined directly in the tumors but the subtypes of infiltrating cells have been studied by detection of specific transcription factors. The transcription factors that specify lineage commitment in T helper cells have started to be deciphered and the generally accepted model considers T-bet and GATA-3 as the master regulators of the so-called Th1 and Th2 differentiation, respectively, with c-Maf as the downstream factor that selectively controls IL-4 gene transcription ,135. TILet al. [TNF-α can be secreted by several types of tumor-infiltrating cells, e.g., fibroblasts, macrophages, NK cells, T cells, and could exert cytotoxic functions on tumor cells . TNF-α et al. , 138-140et al. -145.4.5.Taken together, these results give the impression that TIL are functional at tumor sites. This is in contrast with the results of the T cell function assays described in the next section. One possible explanation is that most anti-tumor TIL are anergic and that only few functional TIL are detected by immunohistochemistry. Another explanation is that the functional TIL detected by immunohistochemistry have been activated outside the tumor, e.g., by antigens derived from Epstein-Barr virus or cytomegalovirus, and have been recently attracted into tumors by chemokines.5.i.e., calcium increase in the cytosol, was poor when TIL from numerous tumors were re-stimulated ex vivo, i.e., tested on untreated and uncultured samples [in vitro for days or weeks, they are usually able to secrete cytokines and kill tumor cells [ex vivo [ex vivo with human TIL.Even the first sign of T cell activation,  samples . Howeveror cells -149. Dur[ex vivo . We summ5.1.T cells isolated from chronic lymphocytic leukemia patients showed impaired immunological synapse formation with leukemic B cells associated with defective actin polymerization . Defecti5.2.MART-1 specific CD8 T cells were isolated with HLA-peptide multimers from melanoma or blood samples and short-term stimulated with peptide-pulsed cells. Eight percent of these TIL contained IFN-γ, whereas 44% of the multimer+ T cells isolated from blood did contain IFN-γ [Anti-melan-Ain IFN-γ . Anti-cyin IFN-γ .How to explain the observations that anti-virus T cells were functional, whereas the anti-tumor cells were not? If the tumor environment is suppressive, why were the anti-virus T cells not sensitive to suppression? One explanation is that anti-virus T cells were residing in the tumor only for a short time. Another explanation is that anti-tumor T cells have been repetitively stimulated by tumor antigens and are therefore more sensitive to immunosuppression.We have tested CD8 and CD4 TIL, which were isolated from melanoma and carcinomatous ascites of the ovary, colon, and pancreas. TIL were stimulated with beads coated with anti-CD3 and anti-CD28 antibodies, which is a strong stimulus. Compared to donor blood T cells, CD8 and CD4 TIL secreted low levels of IFN-γ and TNF-α .5.3.CD8 TIL isolated from renal cell carcinoma, malignant pleural effusions, ovarian and pancreatic carcinoma ascites failed to lyse cells coated with anti-CD3 antibodies in an assay known as a redirected killing assay, whereas CD8 blood T cells were cytolytic ,155. Pre5.4.ex vivo functional assays whereas the reports on signs of TIL activity in situ gave the opposite impression. Because the ex vivo functional assays are rather straightforward, we are tempted to conclude that most TIL are actually dysfunctional. We hope that additional experiments from different groups will help strengthen this conclusion. There is also a need for functional assays with control T cells isolated from non-cancerous tissues such as acute inflammatory sites.Taken together, the few results reported above give the impression that most TIL are dysfunctional in 6.A reasonable hypothesis is that the tumor microenvironment impairs TIL functions. We will focus in this part of the review on the various mechanisms able to decrease TIL efficacy, in particular the mechanisms that could be inhibited by pharmacological methods. We will not discuss the selection of tumor cell variants that no longer present the antigens that are the targets of TIL. This tumor escape mechanism has been described in detail -159.6.1.in vitro or in clinical trials.Several molecules expressed by TIL could down-modulate their activation upon antigen recognition. A more complete set of such molecules is described in 6.1.1.+ TIL isolated from melanoma that also expressed Programmed Death-1 (PD1) produced very low levels of IFN-γ upon stimulation with PMA-Ionomycin, a strong non-specific stimulus [CTLA-4, expressed on T cells, can compete with CD28 for the binding to CD80/86, expressed on antigen-presenting cells and, consequently, prevents recruitment of PKCθ at the synapse and T cell activation . Additiostimulus .http://www.clinicaltrials.gov/). In a phase-3 study, 676 unresectable stage III or IV melanoma patients received either a vaccine containing two gp100 peptides corresponding to antigens expressed by melanoma and normal melanocytes, or Ipilimumab alone, or Ipilimumab plus the gp100 vaccine [in vivo, and by extension, the mechanism of anti-tumor immune activity mediated by CTLA-4 blockade, is an area of active investigation [Anti-CTLA-4-blocking monoclonal antibodies Ipilimumab and Tremelimumab have already been tested in many clinical trials, both in melanoma patients and in patients with other malignancies -175 , are phosphorylated upon ligand binding and, consequently, recruit SHP-1 -182. It te IFN-γ . This dr+ tumor cells or peptide-pulsed blood mononuclear cells, either diminished aptotosis of specific T cells [Blockade of PD-1/PDL-1 interactions seems to prolong survival of T cells and promote their expansion rather than reverse T cell dysfunction. Addition of blocking anti-PD-1 antibodies in cultures of human T cells stimulated with peptide-pulsed dendritic cells increased by 2–3 fold the proliferation of anti-peptide T cells , whereas T cells , or incr T cells . Anti-PD T cells . Another T cells . One dur+ CTLA-4+ CD8 T cells, which were isolated from the liver of hepatitis C-infected patients and stimulated with peptide in the presence of anti-PD-L1 and anti-CTLA-4 blocking antibodies [There is some rationale for combining therapies with different antibodies as blood CD8 T cells from cancer patients can co-express multiple inhibitor receptors, such as PD-1 and Tim-3 . Upon antibodies .6.1.3.The mere detection of inhibitory receptors on TIL, is sometimes considered as a marker of TIL impairment -193, des6.2.Numerous immune cell types have some level of immunosuppressive activity. Among them are Treg, myeloid-derived suppressor cells, mesenchymal stem cells and tumor-associated macrophages. Only the presence of Treg and macrophages has been associated with survival prognosis and will be discussed below.6.2.1.+ T cells with a CD25+FOXP3+ phenotype are generally considered as Treg. In humans, this definition includes regulatory T cells but also activated CD4+ T cells [CD4 T cells , 220. Ot T cells ). Treg c T cells .+CD25+ or CD4+FOXP3+ was correlated with poor survival in renal cell carcinomas, gastric carcinomas, breast carcinomas, ovarian carcinomas, non-small-cell lung carcinomas, melanomas and colon adenocarcinomas [, but the presence of Treg was correlated with a greater survival in nasopharyngeal carcinoma, gastric carcinoma and lymphoma patients [The lack of specificity of these markers can explain the contradictory reports on Treg and survival. The relevance of most of these studies is therefore questionable. Infiltration by CD4rcinomas ,223-232,patients ,233,234.FOXP3 [The most specific marker of human Treg up to now is an epigenetic marker: the demethylation of the first intron of FOXP3 ,220,235.+ regulatory T cells were administered to cancer patients : cyclophosphamide, anti-CD25 monoclonal antibody Daclizumab, and Denileukin diftitox (Ontak®), an engineered protein combining IL-2 and diphtheria toxin [Different treatments aimed at reducing the number of CD25ia toxin -238. Thiper os [+CD25+ T cells was documented only after per os administration. It was also injected i.v. at high doses and induced lymphodepletion, therefore possibly removing a cytokine sink [Cyclophosphamide was injected i.v. at low doses or given per os -241. A tine sink ,243. Meline sink . Eightee+CD25+ T cells. However, Treg are only a subset of CD4+CD25+ T cells. When the demethylation of FOXP3 intron 1 was used to detect Treg frequencies, neither Daclizumab nor Denileukin diftitox was able to halve the frequency of Treg cells [Daclizumab was efficient in depleting transiently CD4eg cells . Adminiseg cells . Furthereg cells . This ef6.2.2.Infiltration of tumor by macrophages was correlated in several malignancies with an increased tumor angiogenesis and poor prognosis -254. Actin vitro human T cell proliferation and production of IFN-γ and IL-2, while it spared the production of Th2 cytokine IL-4 [in vitro proliferation of T cells isolated from patients with multiple myeloma or head and neck cancer [Macrophages and cancer cells can also express the enzyme cyclooxygenase 2 (COX2) which metabolizes arachidonic acid into prostaglandin E2 (PGE2) -262. PGEine IL-4 -265. Thuine IL-4 -262. Notine IL-4 . Drugs ck cancer ,267.Tumor-associated macrophages were associated with a favorable prognosis in cutaneaous melanoma ,268,269.6.3.in vitro.We describe below soluble molecules that can modulate TIL activation and function. A more complete set of molecules is shown in 6.3.1.In vitro, the active forms of TGF-β and IL-10 are classically described as immunosuppressive [www.clinicaltrials.gov). No clinical results have yet been reported. Fresolimumab, another anti-TGF-β blocking antibody, has been tested in systemic sclerosis, but not in cancer patients [pressive -274. Howpatients .6.3.2.Indoleamine Dioxygenase (IDO) is an enzyme that catalyzes the first step in tryptophan catabolism along the kynurenine pathway. Breakdown of tryptophan inside IDO-expressing tumor cells results in the consumption of available tryptophane in the local tumor environment and thus in deprivation of this essential amino-acid for the T cells . The met+ mature dendritic cells with autologous human T cells, frequencies of CD4+CD25+Foxp3+CD127- T cells increased by more than 2-fold. This was prevented by the addition of 1-methyl tryptophan to the co-culture [It has been proposed that tryptophan starvation and presence of kynurenines can convert murine naive conventional CD4 T cells into highly suppressive regulatory CD4 T cells. In turn, regulatory T cells would induce IDO expression in dendritic cells, which can further expand the regulatory T cell compartment . After 6-culture .IDO is overexpressed in a variety of tumors and is upregulated in many cells in response to IFN-γ .In metastatic melanoma patients, the patients with a short survival (mean: 8 months) had all an IDO-expressing tumor, as detected by immunochemistry, whereas only 20% of the long survivors (mean: 262 months) had an IDO-positive tumor . IDO expTwo other enzymes that degrade amino-acids were described to play an immunosuppressive role in cancer: arginase and NO-synthase ,310. Bot6.3.3.The metabolism of tumor cells can generate high amounts of lactic acid, due to up-regulation of glycolytic enzymes and hypoxia at the tumor site . Adding in vitro cytokine production and cytotoxic activity by CD4+ and CD8 T cells [By hydrolyzing ATP, hypoxic tumor cells could release adenosine . Adenosi T cells .6.3.4.Peroxynitrites, which belong to reactive oxygen species, seem to be produced at the tumor site in human prostate carcinoma and in different human carcinoma cell lines ,318. In In humans, monocytes and a subpopulation of neutrophils, described as myeloid derived suppressor cells, were shown to produce reactive oxygen species . A subpo6.3.5.ex vivo re-stimulation. We have attributed the decreased IFN-γ secretion to a reduced mobility of T cell receptors upon trapping in a lattice of glycoproteins clustered by extracellular galectin-3 [We have observed that human CD8 TIL, in contrast with CD8 blood cells, show impaired IFN-γ secretion upon lectin-3 . Indeed,7.7.1.i.e., sugar- or glycan-binding proteins. Galectins share the ability to bind β-galactosides and contain homologous carbohydrate recognition domains (CRD). Their binding affinity for simple β-galactosides, such as disaccharides or trisaccharides, is relatively weak. However, galectin binding to natural glycoconjugate ligands expressed on cell surfaces or in the extracellular matrix is usually of higher affinity, in the submicromolar range. We will discuss below the role in the tumor microenvironment of the sole galectin-1, -3, and -9. Further reading on galectins can be found in reviews by Rabinovich [Galectins belong to a family of lectins, binovich  and Cummbinovich .Galectins have been classified in three structural groups: (a) the prototype galectin-1 with a single CRD, which can form homodimers via non-covalent interactions; (b) the chimeric galectin-3 with a single CRD and a large amino-terminal domain, which contributes to self-aggregation into pentamers; and (c) the tandem-repeat galectins with two different CRD and a 5 to 70 amino-acid linker, e.g., galectin-9.O- and N-glycoproteins. The interactions of galectins and glycoproteins are complex and cannot be interpreted in terms of interactions between a ligand and a receptor. Several factors contribute to high-affinity binding to natural glycoconjugate ligands: the natural multivalency, the oligomeric state of the galectins and the multivalency of their natural glycoconjugate ligands [Galectins are found in the cytosol and in the nucleus where they interact with proteins to regulate growth, cycle progression and apoptosis ,327. Gal ligands .+CD25high cells, possibly Treg cells, was reported to be reversed by an anti-galectin-1 antibody [Galectins participate in both glycoprotein clustering and glycoprotein/galectin lattices, and could therefore modify receptor signaling and increase the surface half-life of some glycoproteins . In tumoantibody . T cell antibody -340.7.2.Sera of melanoma and colon adenocarcinoma patients were reported to contain more galectin-3 than sera of healthy volunteers ,342. Mel7.3.in vitro exposure to galectin-1 induced apoptosis of subsets of CD4lo CD8lo thymocytes [Blood T cells, T cell lines or Jurkat T cells were stimulated in the presence of galectin-1, -3, or -9, usually added in the culture at μM concentrations. Treated cells either entered in apoptosis -349, proymocytes .Galectin-1, -3 and -9 added at μM concentrations also induce apoptosis of prostate carcinoma, melanoma, B-lymphoma and leukemia cells, provided they harbor the adequate glycosylation pattern ,354-357.The apoptosis induced by galectin-1 and galectin-3 seems to be mediated by their interactions with highly glycosylated surface molecules, such as CD43 and CD45, as these molecules co-purified with these galectins and were associated in patches at the T cell surface ,358-360.N-glycans exposed at the cell surface, as shown for murine T cells [We have reported that treatment of TIL with galectin ligand LacNAc boosted their secretion of cytokines upon stimulation. Why do galectin-3 ligands improve TIL function? Our working hypothesis is that TIL have been stimulated by antigen recently, and that the resulting activation of T cells could modify the expression of enzymes of the N-glycosylation pathway and change the structure of  T cells . We surm T cells .Ex vivo treatment of TIL with galectin-3 ligand LacNAC boosted their functions [®, a modified citrus pectin with cytotoxic properties on tumors, was administered by i.v. injection to 24 multiple myeloma patients [In vitro, GCS-100 detached galectin-3 from TIL and boosted cytotoxicity and secretion of different cytokines [®, another modified citrus pectin, was given orally to 10 prostate cancer patients [unctions . We havepatients -368. Sidytokines . Pectasopatients . Seven o®, a galactomannan derived from guar gum, has been shown to bind to galectin-1 [ex vivo treatment of TIL with DAVANAT boosted secretion of cytokines and lytic activity (our own unpublished data). We intend to launch a clinical trial with metastatic melanoma patients where MAGE3.A1 and Na17.A2 peptides, which correspond to tumor-specific antigens expressed by various types of tumor, will be administered together with systemic and local injections of DAVANAT.DAVANATlectin-1 , and to lectin-1 . Short e8.There is now an increasing number of available data on human tumors and TIL to support the scenario of an immunosuppressive tumor microenvironment. The respective contribution of the different immunosuppressive mechanisms remains to be clarified in order to define whether different lymphocyte populations are impaired through different mechanisms, and whether several inhibitory pathways add up in the same cells. Considering the remarkably low toxicity of therapeutic vaccination of cancer patients, provided the target antigen is tumor-specific, every possible effort to improve its efficacy should be done. It was proposed that, in some vaccinated patients, a few anti-vaccine T cells reach metastases, succeed in reversing the immunosuppression, and trigger a broad activation of other anti-tumor T cells that proceed to eliminate the bulk of the tumor cells ,111,120.In the absence of vaccinations, treatments aimed at boosting TIL functions in vaccinated patients could also trigger activation of anti-tumor T cells. The same treatments should also contribute to provide a better tumor microenvironment for the anti-vaccine T cells able to reach the tumor sites.We should keep in mind that boosting TIL function may result in increased secretion of molecules that indirectly promote tumor progression and that simultaneously blocking several surface inhibitory receptors may increase the severity of side-effects.


In vitro assays showed that aberrant expression of miR-155 significantly enhanced GBC cell proliferation and invasion. In conclusion, high miR-155 expression correlates with the aggressive behavior of GBCs, and miR-155 may become a prognostic marker and therapeutic target for GBC.The prognosis of gallbladder cancer (GBC) remains poor despite recent advances in diagnostics and therapeutic strategies. Although the role of microRNAs (miRs) in GBC have not been well documented, miR-155 is known to be associated with inflammation-associated carcinogenesis in various types of cancers. The aim of this study was to investigate the clinical significance of miR-155 expression and the biological functions of miR-155 in GBC. The expression levels of miR-155 in surgically resected GBCs and gallbladders with pancreaticobiliary maljunction (PBM) were assessed by quantitative reverse transcription-polymerase chain reaction. The relationship between the expression levels of miR-155 and clinicopathological features of GBCs was analyzed. Human GBC cell lines were transfected with miR-155 inhibitors or mimics, and the effects on proliferation and invasion were assessed. miR-155 was significantly overexpressed in GBCs when compared with that in gallbladders with PBM (P=0.007) and normal gallbladders (P=0.04). The high expression level of miR-155 in GBCs was significantly associated with the presence of lymph node metastasis (P=0.01) and a poor prognosis (P=0.02).  The prognosis of gallbladder cancer (GBC) remains poor despite recent advances in diagnostic modalities and therapeutic tools. The incidence of GBC is >4,000 cases per year, and the 5-year survival rate of patients with GBC is estimated to be ~12% . Therefode novo’ oncogenesis  is a major risk factor for GBC and is one of the clues used to clarify the mechanisms of carcinogenesis and malignant behavior of GBCs. Reflux of pancreatic juice into the bile duct activates the conversion of lecithin to lysolecithin by phospholipase A2, and concentrated lysolecithin in the gallbladder or the dilated bile duct induces chronic inflammation and damage to biliary epithelium . K-ras aogenesis –9. Thus,microRNAs (miRs) are non-coding RNAs consisting of 18–25 nucleotides, which have been reported to play important roles in the regulation of carcinogenesis and cancer progression as well as homeostasis. Approximately 1500 miRs have been identified to date, and aberrant expression of miRs has been detected in various types of malignancies including breast, colorectal and lung cancers ,11. Althet al in accordance with the manufacturer's protocol. The RNA quantity was measured with an ND-1000 spectrophotometer . Total RNA was checked by a 2100 Bioanalyzer  to determine the RNA quality integrity number and 28s/18s rRNA ratio.Purified RNAs were subjected to miR assay. A stem-loop quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed with a TaqMan MicroRNA Reverse Transcription kit and TaqMan Universal PCR Master Mix II  and Chromo4 real-time PCR detection system  for each sample according to the manufacturer's instructions. cDNAs were placed in triplicate onto a plate in a total volume of 5 μl/well. Briefly, for reverse transcription, the reaction mixtures were incubated at 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, and then maintained at 4°C. PCR amplification was performed at 95°C for 10 min, followed by 40 cycles at 94°C for 15 sec and 60°C for 60 sec. The expression levels of specific miRs were normalized to those of RNA U6 (RNU6B) as the endogenous control.2.Human GBC cell lines G-415 , OCUG-1 , and NOZ  were used in the present study. The G-415 cell line was purchased from the Riken BioRecourse Center through the National Bio-Resource Project of the Ministry of Education, Science, Sports Culture and Technology , and maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS). OCUG-1 and NOZ cell lines were purchased from the Japan Health Science Research Resources Bank , and maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and Williams' E medium with 10% FBS, respectively. Each cell line was cultured at 37°C in a humidified incubator with 5% COTo analyze the expression levels of miR-155 in the GBC cell lines by qRT-PCR, total RNA was extracted from each GBC cell line using a mirVana miRNA Isolation kit (Applied Biosystems) according to the manufacturer's instructions. qRT-PCR was performed using a Chromo4 real-time PCR detection system with TaqMan microRNA assays (Applied Biosystems) as described above.6 cells suspended in 100 μl Nucleofector solution, followed by electroporation. The sequence of the mimic for miR-155 was 5′-UUAAUGCUAAUC GUGAUAGGGGU-3′. The sequence of the inhibitor for miR-155 was 5′-UCCCCTUTCUCGUTTUGCUTTUU-3′. Negative control miR was used as described by Gregory et al according to the manufacturer's protocol. Briefly, 100 pmol of the inhibitors or mimics of miR-155 or a negative control miR was added to 1×10ory et al. Synthes4 cells/well were seeded in 24-well plates containing medium without phenol-red and then incubated at 37°C for 48 and 96 h. At the indicated time, 30 μM PI and 600 μM digitonin were added to each well. After incubation for 30 min at 37°C, the fluorescence intensity was measured by an Infinite 200 multi-well plate reader  with 530-nm excitation and 645-nm emission filters, and the total number of cells was calculated. Each experiment was performed three times in triplicate.Cell proliferation was assessed by a modified propidium iodide (PI) assay as previously described . Briefly5 cells suspended in 250 μl culture medium were seeded into each insert. After 16 h of incubation at 37°C with 5% CO2, the non-invading cells on the surface of the membrane were removed by scrubbing with a cotton swab. The invading cells on the lower surface of the membrane were fixed with 70% ethanol and stained with hematoxylin and eosin. The invading cells in five randomly selected fields were counted under a light microscope at ×100 magnification. Each experiment was performed three times in triplicate. Cell growth of GBC cells used in the invasion assays was not observed after 16 h.Cancer cell invasion was assessed by measuring the movement of cells into an artificial basal membrane in Matrigel-coated Transwell chambers . Each insert had an 8-μm pore-sized membrane coated with 20 μg Matrigel. Briefly, 750 μl culture medium was placed into each well of a 24-well plate, and then 1×10The cut-off value of miR-155 expression was set by the median value of the expression levels, and the lesions were divided into two groups according to high and low expression levels of miR-155. The relationship between the expression level of miR-155 and clinicopathological characteristics, including prognosis, was assessed. The effect of possible prognostic factors on survival was also analyzed. Comparison between two groups was analyzed with the Chi-square test, Mann-Whitney U test, or Cox regression test. Cumulative survival rates were calculated with the Kaplan-Meier method and compared with the log-rank test using JMP version 9.0.2 . Differences were considered significant when the probability value was <0.05.The expression level of miR-155 was examined in 26 GBCs (including 9 with PBM), 13 non-cancerous gallbladders with PBM, and 17 normal gallbladders by qRT-PCR . The expHigh miR-155 expression was significantly associated with the presence of lymph node metastasis (P=0.03) and vessel invasion (P=0.007) . There wThe relationship between the expression level of miR-155 and the disease-specific survival (DSS) rate after surgery was examined. DSS was significantly lower for GBC patients with a high expression level of miR-155  than those with a low miR-155 expression level  (P=0.01) . The preThe relative expression levels of miR-155 to RUN6B expression in G-415, NOZ and OCUG-1 cells were 56.8-, 18.1- and 4.2-fold, respectively . ExpressG-415 (P=0.0016) and OCUG-1 (P=0.0071) cells transfected with miR-155 inhibitors showed significant decreases in cell proliferation, compared with that in the negative controls. On the other hand, G-415 (P=0.0009) and OCUG-1 (P=0.0030) cells transfected with miR-155 mimics showed significant increases in cell proliferation, compared with that in the negative controls . NOZ celIn the invasion assay, the number of invasive cells among the total number of G-415 cells transfected with miR-155 mimics was significantly higher compared with that of the normal control (P=0.034), whereas no difference in invasion was observed in OCUG-1 (P=0.32) and NOZ (P=0.93) cells transfected with mimics. The numbers of invasive cells among G-415, NOZ and OCUG-1 cells transfected with miR-155 inhibitors were significantly lower  compared with those of the normal controls .in vitro. To our knowledge, this is the first report demonstrating aberrant miR-155 expression and its effects on the function of GBC cells.In the present study, we found that: i) miR-155 is upregulated in GBCs irrespective of the presence or absence of PBM, ii)high miR-155 expression is significantly associated with the presence of lymph node metastasis and vessel invasion, and indicates a poorer prognosis for GBC patients, when compared with GBC patients with low miR-155 expression, and iii) upregulation of miR-155 affects the proliferation and invasion of GBC cells One of the clinical problems during management of GBCs is that many GBCs are found at an advanced stage with metastasis at the time of diagnosis, and most early-stage GBCs within the mucosal layer are diagnosed incidentally after cholecystectomy for cholecystolithiasis. Because of the difficulty of the early detection of GBCs, identification of sensitive markers to diagnose early-stage GBCs is urgently needed. Our present study was planned based on the hypothesis that assessment of abnormal regulation of miRs may help us to understand the complicated mechanism of carcinogenesis and address the clinical problems of GBC.et al(Recent reports have analyzed the relationship between the expression level of miR-155 and the prognosis of several types of cancers. Shibuya et al showed tet al. Our preet al(et al(Stable miRs have been recently found in human blood, despite the presence of RNase, and abnormal regulation of miRs has been detected in the serum of patients with breast, esophageal and prostate cancers –24. Heneet al identifiet al,26. Rece al(et al demonstret al(et al(in vitro cell proliferation by targeting the transcriptional regulator, sex-determining region Y box 6. Our present study also demonstrated that aberrant expression levels of miR-155 by transfection of miR-155 inhibitors or mimics, were correlated with proliferation and the invasiveness of GBC cell lines. In particular, G-415 cells with an unmodified high expression level of miR-155 showed marked inhibition or promotion of proliferation and invasion following transfection of miR-155 inhibitors or mimics. These results are in good agreement with the hypothesis that miR-155 is a regulator of proliferation and invasion of GBC cells. The present study suggests the possibility that modulation of the miR-155 level may be applied to the treatment of GBCs, particularly for inhibition of cancer progression such as lymph node invasion.miRs are also expected to become molecular targets of cancer therapy . Garzon et al showed t al(et al found thIn contrast to our expectation, PBM did not affect regulation of the miR-155 level in gallbladder epithelium. This finding suggests that miR-155 is not involved in the early stage of multistep carcinogenesis induced by inflammation, the ‘hyperplasia-to-atypical hyperplasia-to-carcinoma sequence’. Furthermore, it appears that the penetration frequency of miR-155 is too low to be a candidate for a general factor involved in carcinogenesis. miR-155 may be overexpressed in the process in which established cancers acquire invasive character.Evaluation of GBCs as well as gallbladders with PBM by microarray may reveal the association of miRs with multistep carcinogenesis under chronic inflammation. miRs are characterized by their binding to the incomplete complementary sites of their targets and allowing mismatched G-U base pairing. However, a single gene target is regulated by several miRs . TherefoIn conclusion, high miR-155 expression correlates with the aggressive behavior of GBCs, and miR-155 may become a prognostic marker and therapeutic target for GBC.


Familial Mediterranean fever (FMF) is associated with variations in the MEFV gene resulting in proteolytic activation of IL-1β through the inflammasome complex. There is no established treatment available for those resistant or intolerant to standard of care colchicine treatment. Canakinumab, a fully human selective anti-IL-1β monoclonal antibody with a half-life of ~4-weeks binds to human IL-1β and neutralizes its proinflammatory effects.To evaluate the efficacy and safety of canakinumab in adolescents and adults with FMF who are resistant or intolerant to colchicine.FMF patients with ≥1 attack/month in the preceding 3-months despite the highest tolerated colchicine dose were eligible to enter a 30-day run-in period. Those with an attack in the run-in period advanced to a 3-month treatment period to receive canakinumab 150mg sc every 4-weeks starting at the next attack in the following month. Patients then followed-up for up to 2 months or until the next attack. Attacks were confirmed by presence of fever, serositis, and elevated CRP. Primary efficacy outcome was the proportion of patients with ≥50% reduction in time-adjusted attack frequency in the treatment vs pre-treatment periods. Secondary objectives included the percent of patients with no attacks in the treatment period, time to next attack after the last canakinumab dose, and changes in the quality of life by SF-36. Safety was assessed by AEs and laboratory values at each visit.run-in and 9  entered treatment periods. Only 1 patient had an attack (peritonitis) during the treatment period and all had a ≥50% reduction in their time-adjusted pre-treatment attack rate. Median baseline elevated CRP (58mg/L) and serum AA (162mg/L) levels normalized  by Day 8 and remained low throughout the study. The Physical and Mental Component scores of the SF-36 improved from a median baseline of 32 and 38 to 81 and 78 at Day 8 respectively, and continued to improve throughout the treatment period. Five patients had an attack in the follow-up period, which occurred a median 71 days (31-78 days) from the last canakinumab dose. Compared to baseline, the physician and patient global assessment of FMF control improved with treatment with overall the response to treatment reported as Very Good by both physicians (9/9) and patients (7/9) at study end. Eight patients reported at least one adverse event (AE) with headache (n=4) and upper respiratory tract infection (n=2) being the only AEs reported in more than 1 patient. No one discontinued early from the trial.Thirteen patients enrolled in the In this pilot study, canakinumab was found to be effective at controlling the attack recurrence in FMF patients resistant or intolerant to colchicine. AEs were similar to previous canakinumab trials and were managable. Further studies are warranted to explore the role of canakinumab in the treatment of FMF.A. Gül Grant / Research Support from: Novartis, Xoma, Servier, Consultant for: Novartis, Xoma, H. Özdoğan Grant / Research Support from: Novartis, Consultant for: Novartis, Ö. Kasapçopur Grant / Research Support from: Novartis, Consultant for: Novartis, B. Erer: None Declared, S. Uğurlu: None Declared, N. Davis Employee of: Novartis, S. Sevgi Employee of: Novartis


Salmonella Typhi, Salmonella Typhimurium and Vibrio cholerae.This study provides information on the antibacterial effect of copper against the water-borne pathogens t tests.Suspensions of each pathogen were kept in water within a traditional copper vessel at 30°C for 24 h. Samples were withdrawn, diluted and plated onto suitable growth media. Conventional enumeration of healthy (uninjured) bacteria was carried out using standard aerobic incubation conditions. Additionally, reactive oxygen species-neutralised (ROS-n) conditions were achieved by adding the peroxide scavenger sodium pyruvate to the medium with anaerobic incubation, to enumerate uninjured (ROS-insensitive) and injured (ROS-sensitive) bacteria. Differences between log-transformed means of conventional (aerobic) and ROS-n counts were statistically evaluated using Overall, all three pathogens were inactivated by storage in copper vessels for 24 h. However, for shorter-term incubation (4-12 h), higher counts were observed under ROS-n conditions than under aerobic conditions, which demonstrate the presence of substantial numbers of sub-lethally injured cells prior to their complete inactivation.The present study has for the first time confirmed that these bacterial pathogens are inactivated by storage in a copper vessel within 24 h. However, it has also demonstrated that it is necessary to account for short-term sub-lethal injury, manifest as ROS-sensitivity, in order to more fully understand the process. This has important practical implications in terms of the time required to store water within a copper vessel to completely inactivate these bacteria and thereby remove the risk of water-borne disease transmission by this route. According to the World Health Organization, an estimated 4.1% of the total global burden of disease is contributed by diarrhoeal illness: around 88% of that burden is due to unsafe water supply, sanitation and hygiene, with children in developing countries being the most common victims -4.Salmonella enterica is of considerable significance, with approximately 22 million cases of enteric fever caused by Salmonella Typhi in 2002  for V. cholerae. The culture was then centrifuged at 5300 × g for 5 min at 5°C and rinsed twice with 0.85% NaCl to remove all traces of growth medium. The pellet was then suspended in the same volume of sterile distilled water and then diluted to 1:100 for experimental purposes, i.e 100 ml of bacterial suspension was added to 10 litres of sterile distilled water at pH 7.0 for S. Typhi, S. Typhimurium and 7.5 for V. cholerae to give a to give a final density of 106 -107 colony forming units per millilitre (CFU mL-1) at 0 h.ironment . This apS. Typhi and S. Typhimurium and to pH 7.5 for V. cholerae. Copper vessels (12 litre capacity), obtained from the local market in Chandigarh were disinfected, scrubbed and rinsed thoroughly to remove any adherent contamination from the inner surface. In order to mimic the rural setting in a laboratory by keeping the cleansing and disinfecting step simple, easy and cost-effective, the present method of rinsing with water, disinfecting and rinsing thoroughly three times with several litres of sterile distilled water each time before use was carried out . Colonies were counted following the appropriate incubation period and the data expressed as CFU ml10CFU ml-1 with 95% confidence limits. 95% confidence limits were used for error bars in the graphs. Differences between log-transformed means of conventional (aerobic) and ROS-n counts were statistically evaluated using t tests. The t and p value for two sets of log values at the same time point was calculated using the t test: paired two sample for means function in Microsoft Excel spreadsheet.Colony counts are expressed as mean logS. Typhi 733 and S. Typhi Ty2 in water stored in a copper vessel for up to 24 h, enumerated under normal aerobic conditions and ROS-n conditions. Counts for both strains fell below the minimum detection limit by 10 h with the degree of sub-lethal injury, demonstrated in terms of the difference between aerobic and ROS-n counts, being more pronounced for S. Typhi Ty2 at 48 h. This difference was statistically significant at 4 h  and beyond. A statistically significant difference between aerobic and ROS-n counts of S. Typhi 733 was also observed at 4 h . No counts were detected for both strains after 8 h under aerobic conditions and ROS-n conditions.Figure S. Typhimurium 98 and S. Typhimurium 1251 , indicating sub-lethal injury to both strains. Counts for both strains of S. Typhimurium fell below the minimum detection limit after 12 h under aerobic and ROS-n conditions.1 Figure  and 2b wV. cholerae 3906 and V. cholerae O1 there was minimal change in counts over the first 4 h of storage, followed by a more rapid decrease with both aerobic and ROS-n counts falling below minimum detection limit after 10 h . Such differences represent a substantial number of sub-lethally injured cells, e.g. at 10 h the counts were 5 × 102 CFU ml-1 for V. cholerae 3906 under aerobic conditions and 1.5 × 104 CFU ml-1 under ROS-n conditions, with equivalent counts for V. cholerae O1 of 5 × 102 CFU ml-1 and 1.0 × 104 CFU ml-1.For h Figure  and 3b. Overall, all test strains were completely inactivated (with no counts obtained under aerobic or ROS-n conditions) by storage in water kept in a copper vessel for 24 h, though all demonstrated sub-lethal injury (sensitivity to aerobic enumeration conditions) during shorter-term exposure. In contrast, storage of the same strains in water maintained in glass vessels resulted in no substantial change in aerobic or ROS-n counts up to 24 h (control data shown in Figures Despite global initiatives in water and sanitation, enteric fever and cholera remain major public health problems in many developing countries ,23, primS. Typhi, S. Typhimurium and V. cholerae. We have also demonstrated that injury is caused to the bacteria during short-term storage, and that this must be taken into account in order to obtain a comprehensive view of the antibacterial process, since ROS-sensitive bacteria are not detected by conventional procedures, and would therefore be missed during counting using standard laboratory methods and media. This is a significant finding as the sub-lethally injured microorganisms maybe as important as their healthy counterparts as they could have the potential to resuscitate in a favourable environment [V. cholerae and V. vulnificus is affected by environmental conditions [V. vulnificus [V. vulnificus [V. parahaemolyticus [The present study has established the antibacterial effect of copper against some of the most important water-borne pathogens of developing nations, namely ironment . The frenditions  and it hnditions . Howeverlnificus  provideslnificus  and V. polyticus  through S. Typhi, S. Typhimurium and V. cholerae by > 105 CFU ml-1, dropping below the minimum detection limit by this time. Consequently, 24 h storage is recommended in preference to shorter-term storage, e.g. overnight. Our previous studies have demonstrated leaching of copper ions when water is stored at 35°C at pH 7.0. The total dissolved copper was measured using a Perkin-Elmer Analyst atomic absorption spectrophotometer, Perkin-Elmer, USA at the Post Graduate Institute of Medical Sciences, Chandigarh [The findings of the present study are significant at the practical level, since they provide information that extends the findings of earlier studies, based on conventional aerobic counts ,15. Whilandigarh . ClearlyEffective point-of-use water disinfection methods are required to reduce the incidence of diarrhoeal diseases in developing nations such as India. We have demonstrated the value of using traditional copper storage vessels to inactivate the water-borne pathogens responsible for typhoid fever and cholera, while also highlighting the occurrence of sub-lethal injury during storage for periods of less than 24 h. The application of improved methods for water storage at the household level are likely to have a significant impact on the overall health of the community ,26.The authors declare that they have no competing interests.RR and SC conceived the research topic. RS designed and conducted the laboratory experiments and interpreted the results. Data analysis was carried out by RR and RS. All authors co-drafted the manuscript and approved the final text. RS is guarantor of the paper.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/204/prepub


The removal of high-abundance proteins from plasma is an efficient approach to investigating flow-through proteins for biomarker discovery studies. Most depletion methods are based on multiple immunoaffinity methods available commercially including LC columns and spin columns. Despite its usefulness, high-abundance depletion has an intrinsic problem, the sponge effect, which should be assessed during depletion experiments. Concurrently, the yield of depletion of high-abundance proteins must be monitored during the use of the depletion column. To date, there is no reasonable technique for measuring the recovery of flow-through proteins after depletion and assessing the capacity for capture of high-abundance proteins.In this study, we developed a method of measuring recovery yields of a multiple affinity removal system column easily and rapidly using enhanced green fluorescence protein as an indicator of flow-through proteins. Also, we monitored the capture efficiency through depletion of a high-abundance protein, albumin labeled with fluorescein isothiocyanate.This simple method can be applied easily to common high-abundance protein depletion methods, effectively reducing experimental variations in biomarker discovery studies. Extracellular body fluids that contain plasma and serum are the most valuable sources for biomarker discovery and application, but useful protein biomarkers exist at low concentrations (ng/ml or lower) in blood; approximately 30 high-abundance proteins constitute ~99% of the total protein mass in human plasma, rendering the detection of low-abundance proteins difficult. Several methods have been developed to deplete high-abundance proteins from blood, most of which are based on the immunoaffinity of antibodies toward high-abundance proteins, for example, multiple affinity removal system  columns which can remove several specific high-abundance proteins -5, but sIn fact, the reproducibility of the removal and recovery of flow-through proteins is a critical aspect of high-abundance protein depletion and although Veronica et al. reported that in a triplicate 2-DE experiment, the coefficient of variation (C.V.) of flow-through and bound fractions by MARS depletion was 5.31% and 4.12%, respectively, 3 depletions were not sufficient to validate the reproducibility with regard to column life  . ChengjiE. coli protein expression system and purified, and a known amount of EGFP was spiked into a clinical sample, such as plasma or serum. During the depletion of high-abundance proteins from plasma or serum, EGFP is an indicator of flow-through proteins out of the MARS depletion column. EGFP exists stably in plasma, and EGFP monitoring by UV absorbance at 488 nm (UV488 nm) provides quality monitoring data on reproducibility and recovery during depletion. In addition, one must track not only the flow-through proteins but also the high-abundance proteins that bind the antibody-based depletion column. According to data from Agilent, buffer B, which is used in the elution step, is based on urea; thus, Agilent has specified that column performance is maintained up to 200 uses. Thus, during repeated use, depletion columns capture fewer high-abundance proteins, and the life of the column varies, depending on handling. Further, column performance cannot be monitored online. Routine SDS-PAGE and Western blot or ELISA can be performed to determine the efficacy of depletion. These processes, however, take time and are costly.In this study, EGFP was expressed in an 488 nm during an HPLC run. Thus, we developed a convenient method for measuring: 1. the recovery of flow-through proteins by monitoring EGFP and 2. the removal of high-abundance proteins using FITC-HSA at UV488 nm during depletion. This online monitoring system conveniently tracks the recovery and performance of all depletion systems that are run by HPLC, including the MARS depletion column. In biomarker discovery or verification steps, periodical online monitoring using EGFP or FITC-HSA assesses the quality of depletion runs and identifies unsuitable cases from the depletion runs, which can reduce experimental variation.In this study, we labeled human serum albumin (HSA), the most abundant protein in plasma, with fluorescein isothiocyanate (FITC) to monitor the capacity of a depletion column. A known amount of FITC-labeled HSA (FITC-HSA) was spiked into depleted plasma or depleted serum as an indicator of high-abundance proteins. Because FITC also has a maximum excitation at 488 nm, we measured the depletion efficiency using EGFP for flow-through proteins and FITC-HSA for high-abundance proteins by online monitoring at UVMARS columns (4.6 mm × 10 mm), solution A, and solution B (Product No. 5185-5985) were provided from Koreabiomics, a distributor for Agilent in Korea. There are limited data on solutions A and B, which are based on water with sodium azide and urea, respectively. For FITC labeling, fluorescein isothiocyanate  and human serum albumin  were purchased from Sigma. PCR primers for EGFP were synthesized by CosmoGentech . LB media powder was purchased from Biopure . Urea for 2DE-PAGE was purchased from Amresco , and thiourea was obtained from GE Healthcare . Sodium carbonate for FITC labeling was purchased from Amresco (Code: 0585-500G), and sodium thiosulfate was purchased from Sigma (Cat. No.: S-7026). Other reagents that are not mentioned here were obtained from Sigma.g at 4°C for 10 minutes to remove debris. The centrifuged plasma was stored in 50-μl aliquots at -80°C until use. For the depletion experiments, the plasma was thawed at 4°C.Human plasma samples were provided by Seoul National University Hospital. All individuals provided informed consent before being enrolled in this study, in accordance with the approved protocol of the Institutional Review Board at Seoul National University Hospital. To reduce sampling bias due to individual variations, 100-μl plasma samples from 10 healthy persons  were pooled and centrifuged at 22,250 http://www.lablife.org. The entire EGFP coding sequence (residues 1-231) was generated by PCR using the vector pEGFP N1  with the sense primer 5'- GACAAGCTTATATGGTGAGCA-3' and the antisense primer 5'-GCCGGGATCACTCTCGAGCAC-3'. The underlined italic letters denote HindIII and XhoI restriction sites, respectively, and the bold letters are additional bases that generated an open-reading frame. After digestion with each restriction enzyme, gel extraction was performed, followed by ligation into the prepared pET 24a (+) vector .The sequence of EGFP was obtained from the Lablife website pET24a(+)-EGFP-His was transformed into BL21 RIL codon-plus cells (DE3) (Novagen), and overnight culture seeds of BL21-CodonPlus (DE3)-RIL that harbored the EGFP DNA construct (5 ml) were used to inoculate 200 ml LB medium, containing kanamycin (30 μg/ml), and grown overnight at 37°C. When the optical density of the cells at 600 nm reached 1.0, protein expression was induced by adding 0.5 mM isopropyl β-d-thiogalactopyranoside (IPTG) at 21°C.g for 10 min. Cell pellets were resuspended in ice-cold 20 mM Tris-HCl (pH 7.8), containing 200 mM NaCl, 1 mM β-mercaptoethanol (βME), and protease inhibitor cocktail . Resuspended cells were lysed by ultrasonication. Cell supernatants were generated by centrifugation at 15,000 g for 20 min at 4°C. EGFP was purified by Ni-NTA purification. Amounts of EGFP were measured by BCA analysis and UV488 nm absorbance at the same concentrations.After overnight induction, cells were harvested by centrifugation at 5,000 R2.To construct a standard curve for EGFP, EGFP was serially diluted from 0 to 0.6 mg/ml  in MARS buffer A . Two hundred microliters of each diluted sample  was transferred to a 96-well plate  and read from 400 nm to 600 nm on an ELISA reader ; the standard curve was generated from known amounts of EGFP, and the observed absorbance at 488 nm was calculated by linear regression and One hundred micrograms of HSA (1.5 μmol)  was dissolved in 20 ml of 100 mM sodium carbonate (pH 9.3), and 1.5 mg of FITC (3.85 μmol) (M.W: 389.38 Da) was dissolved in 500 ml dimethyl sulfoxide . The solutions were mixed at a 1:2.56 molar ratio (HSA: FITC = 1.5 μmol: 3.85 μmol) and incubated overnight at 4°C.Solutions of FITC-HSA and unlabeled FITC were concentrated using a 5-kDa cutoff Centricon filter , dialyzed in Slide-A-Lyzer dialysis cassettes  in 1 L PBS 5 times , and reconcentrated on a 5-kDa cutoff Centricon filter (Millipore) to remove unlabeled FITC. FITC-HSA was concentrated to 500 μl and purified on a Superdex S-200 gel filtration column by AKTA FPLC  to enhance the purity. A 30-cm Tricon column  was used at a flow rate of 0.3 ml/min. PBS was used as the buffer for size-exclusion chromatography. After gel filtration, the eluted fraction of pure FITC-HSA was pooled, concentrated to 1 mg/ml, and stored in the dark at 4°C until use.To deplete plasma using the MARS column, plasma was diluted 5-fold with MARS buffer A and filtered on a 0.22-μm Ultrafree-MC Durapore centrifugal filter . Plasma only, EGFP-spiked plasma, and EGFP-only samples were applied to a MARS column on an LC-10AT HPLC system , as described below. The sample loop volume of HPLC was set to 100 μl, wherein 58 μl of 5-fold diluted plasma  and 42 μl EGFP  for EGFP-spiked plasma or 58 μl MARS buffer A and 42 μl EGFP  for EGFP-only were injected into the MARS column.The total LC run time of 45 min comprised the following sequence: 100% MARS buffer A at a flow rate of 0.7 ml/min for an initial 15 min, sample injection, a 15-min wash, 100% MARS buffer B at a flow rate of 1.0 ml/min for 5 min, and 100% MARS buffer A at a flow rate of 0.7 ml/min for 10 min. The UV detector was set to 280 nm for the injection of plasma only or 488 nm for the injection of EGFP-spiked plasma or EGFP-only samples, and the eluted fractions were collected in 500 μl. The flow-through and bound fractions were eluted in 5  and 10 vials , respectively, for which the 2 eluted fractions were pooled, respectively. The 2 fractions were reduced to 1 ml on a Centricon. After determining the concentration of the fractions by BCA assay, the fractions was subjected to Western blot, 1-DE, or 2-DE PAGE. The resulting UV chromatograms were analyzed to determine peak areas and retention times.To determine whether FITC-HSA, as an indicator of high-abundance proteins was separable on a MARS column, 600 μg FITC-HSA was injected into a MARS column in triplicate while the UV detector was set to 488 nm. In addition, the depletion of FITC-HSA from plasma was examined, wherein 600 μg FITC-HSA was spiked into HSA-depleted plasma to simulate HSA-undepleted plasma. The HSA-depleted plasma that was spiked with FITC-HSA was injected into a MARS column in triplicate with the UV detector set to 488 nm. The resulting UV chromatograms were analyzed to calculate peak areas and retention times.For plasma depletion, the volumes of the unbound fraction containing EGFP and the bound fraction were 2.5 ml and 5.0 ml, respectively. Each fraction was pooled and concentrated to 1.0 ml on a Nanosep 3K centrifugal filter . Next, 200 μl of the concentrated samples was transferred to 96-well plates  to measure UV absorbance at 488 nm on an ELISA reader, and the UV absorbance was extrapolated on the standard curve to determine EGFP concentration.After plasma depletion, bound fractions that contained high-abundance proteins were pooled and buffer-exchanged with 5 ml of rehydration buffer  pharmalyte (pH 3-10)) 5 times to remove any traces of buffer B. After protein concentration was determined by Bradford assay, 30 μg of bound fraction was subjected to IEF using immobilized pH gradient (IPG) strips , as described . BrieflyGel electrophoresis in the second dimension was performed using a standard SDS-PAGE protocol. After SDS-PAGE, protein spots were visualized by silver staining as described . BrieflyFor Western blot analysis, the concentrated bound fraction in MARS buffer B was buffer-exchanged with 1 ml MARS buffer A 10 times on a Nanosep 3K centrifugal filter; MARS buffer B reacts with BCA solution to generate color. After BCA analysis, 5 μg was separated on 12% SDS-PAGE gels and transferred to PVDF membranes at 100 V  for 1 hr at 4°C.The membranes were blocked with 5% (w/v) BSA, 0.1% Tween 20 in TBS buffer for 2 hr at room temperature and incubated with primary antibodies on a shaker at 4°C overnight. The primary antibodies were mouse anti-human transferrin , mouse anti-human alpha-1-antitrypsin , mouse anti-human haptoglobin , and rabbit anti-his probe .t-test.After being washed in 0.5% Tween 20 in TBS for 10 min 5 times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies  at 1:5000 for 1 hr at room temperature. Signals were detected using ECL Plus Western Blotting Detection Reagents  on an LAS-4000 Image analyzer . Signals were analyzed quantitatively using image analysis software . All results were expressed as means and analyzed statistically by Student's This study is divided into 4 sections, as described in Figure E. coli strain. The yield of EGFP was approximately 50 mg/L in LB media, and the purity was high, as shown by SDS-PAGE  codon plus 280 and OD495 of FITC-HSA and FITC alone were measured; the labeling of HSA with FITC was estimated as described in the FITC labeling protocol [To prepare FITC-HSA, HSA was labeled with FITC at a ratio of 1:2.56, and the labeling efficiency was measured as described in Additional file protocol . ConsequR2 = 0.9988  versus UV absorbance at 488 nm Figure . EGFP co8 Figure . The limTo examine the reproducibility of EGFP as a flow-through indicator in plasma, depletion experiments were performed 6 consecutive times, wherein 58 μl of diluted plasma (2.32 mg), spiked with 42 μl EGFP (300 μg), was applied onto the MARS column. The chromatograms for the 6 runs were reproducible with regard to retention time and UV intensity for EGFP-spiked plasma Figure . The coeA high-abundance protein fraction (30 μg) was subjected to 2-DE and visualized by silver staining. In the 2-DE image, 6 high-abundance proteins were observed Figure , consistNext, we examined the efficacy of EGFP in MARS column depletion by analyzing the bound and unbound fractions of EGFP-spiked plasma. We assessed the efficacy using Western blot by examining whether the MARS column depleted representative high-abundance proteins, such as α1-antitrypsin, haptoglobin, and transferrin, but it did not deplete flow-through proteins, such as His-tagged EGFP Figure . Three . Injection samples were prepared identically in the 6 repeat experiments as in the previous section (300 μg of EGFP and 2.62 mg of plasma protein in 100 μl). EGFP-spiked plasma was injected into the MARS column every 20th run from the 20th to 200th run, and the resulting 10 chromatograms were overlain, as shown in Figure In these UV chromatograms, the peak of unbound fractions (flow-through proteins) appeared at 17 min, and that of the bound fractions (high-abundance proteins) appeared at 35 min in all 6 batches. The C.V. for the peak area and retention time was 8.5% and 0.25%, respectively, at 17 min (EGFP + other flow-through proteins) during EGFP-spiked plasma depletion Table . All runWe examined the recovery of EGFP after the depletion runs. The amount of EGFP that was spiked into the plasma sample was adjusted to 300 μg. The volumes of the EGFP-containing unbound fraction and bound fraction were 2.5 ml  and 5.0 ml  after depletion, respectively. The volumes of the 2 fractions were reduced to 1 ml by Centricon, and the UV absorbance at 488 nm was measured using 200 μl of each concentrated sample to estimate the concentration of EGFP, based on the standard curve ; the C.V. of the fluorescence intensities for the 3 FITC-HSA bands was 5.2%  and CFP (blue), instead of FITC, might increase the efficiency of monitoring the depletion of high-abundance proteins.E. coli expression system, and purified by NI-NTA affinity easily. A 1-L culture of E. coli can provide 50 mg of purified EGFP (data was not shown), which can be used at least 150 times (300 μg per reaction); these steps cost under 300 USD , and EGFP preparation takes a week at most. If EGFP can be supplied as a purified solution form or powder, more time can be saved. The price of FITC powder (1 g) is approximately 200 USD, which can label 172 g HSA to be used at least 280,000 times (600 μg per reaction). HSA powder can be purchased readily and costs approximately 280 USD.EGFP can be cloned, expressed in an FITC-HSA takes most 4 days to prepare, and using EGFP and FITC-HSA renders the monitoring of depletion processes in real time possible. In contrast, if an ELISA kit or Western blot assay that has high sensitivity but is not online or in real time is used to monitor depletion, the initial costs  might be higher, and more time will be spent for post-monitoring of depletion process. With regard to convenience and cost, EGFP and FITC have an immeasurable advantage.The percentage of EGFP that was spiked into plasma is 11.4% (300 μg EGFP in 2.62 mg of the plasma and EGFP mixture), the level of middle abundance proteins in plasma. Since it does not require a mass spectrometer, EGFP-spiked plasma can be used periodically for quality monitoring. In addition, this quantity of EGFP will not interfere significantly with the MS analysis of low-abundance plasma proteins. However, it will be recommended that EGFP would be used to monitor flow-through proteins during depletion rather than low-abundance proteins.Based on this study, we recommend monitoring the efficiency of depletion during the warranted use by the column supplier, using EGFP and FITC-HSA to indicate flow-through proteins and high-abundance proteins, respectively. These indicators can also be used for online monitoring that differentiates normal and abnormal depletions to allow one to remove poor batches or replace the MARS column, as described in Additional file We adopted EGFP as a flow-through protein indicator and FITC-HSA as a high-abundance protein indicator to monitor the recovery and efficiency during depletion of high-abundance proteins from plasma or serum. This system can also be applied to other high-abundance proteins depletion systems. EGFP can be generated easily from cloned EGFP, and FITC-HSA is also readily obtainable. HPLC systems are normally equipped with a UV detector that can monitor UV absorbance of EGFP and FITC-HSA, obviating the need to determine the recovery and efficiency postexperimentally using expensive antibody-based tools, such as ELISA and Western blot. One can determine the efficiency of recovery and reproducibility online during depletion conveniently in this system.Trivial amounts of FITC-HSA were not captured, possibly due to the blocking of antibody epitopes by the FITC-labeled moiety, but these constant unbinding of FITC-HSA were small and taking into these fact into consideration would make analysis more accountable. To overcome this phenomenon, we are attempting to fuse fluorescent proteins with serum albumin, transferrin, and antitrypsin as additional indicators of high-abundance proteins. Although we have used only the MARS column as a model system, our results suggest that the 2 indicator proteins EGFP and FITC-labeled albumin can be used conveniently to monitor the recovery and efficiency of depletion from plasma or serum using other types of immunoaffinity columns. In biomarker discovery, this process maintains the quality in hundreds of plasma depletion runs.The authors declare that they have no competing interests.KK and JY collected clinical plasma samples, performed the proteomics experiments, and wrote the manuscript. HK and BK performed liquid chromatography purification. HM analyzed the MS data statistics. HGY designed the clinical experiments and oversaw the IRB approval procedure. YK supervised the work, provided useful suggestions to improve performance, and revised the manuscript. All authors have read and approved the manuscript.Supplementary data. Four supplementary figures and one table were included.Click here for file


Streptococcus suis is a major swine pathogen and important zoonotic agent causing mainly septicemia and meningitis. However, the mechanisms involved in host innate and adaptive immune responses toward S. suis as well as the mechanisms used by S. suis to subvert these responses are unknown. Here, and for the first time, the ability of S. suis to interact with bone marrow-derived swine dendritic cells (DCs) was evaluated. In addition, the role of S. suis capsular polysaccharide in modulation of DC functions was also assessed. Well encapsulated S. suis was relatively resistant to phagocytosis, but it increased the relative expression of Toll-like receptors 2 and 6 and triggered the release of several cytokines by DCs, including IL-1β, IL-6, IL-8, IL-12p40 and TNF-α. The capsular polysaccharide was shown to interfere with DC phagocytosis; however, once internalized, S. suis was readily destroyed by DCs independently of the presence of the capsular polysaccharide. Cell wall components were mainly responsible for DC activation, since the capsular polysaccharide-negative mutant induced higher cytokine levels than the wild-type strain. The capsular polysaccharide also interfered with the expression of the co-stimulatory molecules CD80/86 and MHC-II on DCs. To conclude, our results show for the first time that S. suis interacts with swine origin DCs and suggest that these cells might play a role in the development of host innate and adaptive immunity during an infection with S. suis serotype 2. Streptococcus suis is a major swine pathogen associated mainly with meningitis, although other pathologies have also been described such as septicemia with sudden death, endocarditis, arthritis, and pneumonia [S. suis disease in humans has been considered as rare and only affecting people working with pigs or pork by-products. However, with a rising incidence in humans over the last years, S. suis is now considered as an important emerging zoonotic agent, especially in Asian countries, where S. suis has recently been identified as the leading cause of adult meningitis in Vietnam, the second in Thailand, and the third in Hong Kong. In 2005, an important outbreak occurred in China and resulted in 200 human cases with a fatality rate near 20% [S. suis is mainly responsible for meningitis, septicemia and streptococcal toxic shock-like syndrome [neumonia . Among 3neumonia . Until rnear 20% . In humasyndrome ,3,4.S. suis infection is still not completely understood and, to date, attempts to control the infection are hampered by the lack of an effective vaccine. The mechanisms involved in the host innate and adaptive immune responses toward S. suis as well as those used by S. suis to subvert these responses are unknown. Several virulence factors have been proposed to be involved in the pathogenesis of S. suis infection [S. suis interactions with host cells, such as endothelial cells, epithelial cells, neutrophils and monocytes/macrophages [Despite the increasing number of studies, the pathogenesis of the nfection . Among tnfection ,6,7 and nfection . In factnfection . Among snfection ,9. The srophages ,5.Dendritic cells (DCs) are powerful antigen presenting cells that initiate the immune response against pathogens, and interactions between DCs and pathogens can strongly influence the outcome of a disease. After the capture of antigens, DCs undergo a complex maturation process, noticeable by the release of cytokines and the increased expression of co-stimulatory molecules. Mature DCs then migrate to the adjacent lymphoid organs where they activate T cells . Thus, DStreptococcus pneumoniae [DCs express a wide variety of pattern-recognition receptors (PRRs) that enable them to detect the presence of several pathogens through the recognition of pathogen-associated molecular patterns (PAMPs). Among these PRRs, Toll-like receptors (TLRs) are important for the recognition of pathogens and the initiation of the immune response as well as the shaping of adaptive immunity . Differeeumoniae ,14.S. suis to interact with DCs and to induce their maturation and activation. We also examined the contribution of S. suis capsular polysaccharide on these interactions. To our knowledge, this is the first study concerning in vitro cultured porcine DC interactions with a whole live bacterial swine pathogen.In the present study, we used porcine bone marrow-derived DCs to investigate the capacity of S. suis serotype 2 virulent suilysin-positive strain 31533, originally isolated from a case of porcine meningitis, and its isogenic non-encapsulated mutant B218 were used. These strains were already used in previous studies [S. suis strains were grown as previously described [S. suis-DCs interaction studies, isolated colonies were used as inocula for THB, which was incubated 8 h at 37°C with shaking. Working cultures were obtained by inoculating 10 μL of a 10-3 dilution of these cultures in 30 mL of THB and incubating for 16 h at 37°C with shaking. Bacteria were washed twice in phosphate-buffered saline  and were appropriately diluted in complete cell culture medium for the experiments. The number of CFU/mL in the final suspension was determined by plating samples onto THA using Autoplate® 4000 .The  studies . S. suisescribed  using eiMycoplasma hyopneumoniae and clinical disease related to porcine circovirus. The herd did not have any episode of acute disease related to S. suis when the samples were taken. All experiments involving animals were conducted in accordance with the guidelines and policies of the Canadian Council on Animal Care and the principles set forth in the Guide for the Care and Use of Laboratory Animals by the Animal Welfare Committee of the Université de Montréal.Cells were obtained from 6-8 weeks old SPF piglets. The animals originated from a herd free of major important diseases such as porcine reproductive and respiratory syndrome (PRRS), enzootic pneumonia due to 6 cells/plate) were cultured in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-Glutamine, 10 mM HEPES and 100 U/mL Penicillin-Streptomycin. All reagents were from Gibco . Complete medium was complemented with 100 ng/mL of porcine recombinant GM-CSF . Cells were cultured for eight days at 37°C in a 5% CO2 incubator and were fed on days 3 and 6. On day 8, cells were harvested, washed, and used as immature DCs for the studies. DC phenotype and purity was confirmed by FACS as described below.Bone marrow-derived DCs were produced according to a technique described elsewhere ,19. BrieS. suis specific antibodies, using a strain-specific ELISA as previously described [2. After incubation, penicillin G (5 μg/mL) and gentamicin (100 μg/mL)  were added into the wells for 1 h to kill extracellular bacteria. Supernatant controls were taken in every test to confirm that extracellular bacteria were efficiently killed by the antibiotics. After antibiotic treatment, cells were washed three times, and sterile water was added to lyse the cells. To ensure complete cell lysis, cells were disrupted by scraping the bottom of the well and by vigorous pipetting. Viable intracellular bacteria were determined by quantitative plating of serial dilutions of the lysates onto THB agar. For intracellular survival studies, an internalization assay was performed as described above, except that after a 60 min initial bacterial-cell contact, gentamycin-penicillin were added and the treatment was lengthened for different times up to 5 h. Cells were then processed as described above and bacteria counted. Results come from at least three independent experiments.Bacteria were either non-opsonized or pre-opsonized using 20% fresh complete normal pig serum in PBS. Serum was negative for S. suis wild-type or its non-encapsulated mutant strain (MOI:1). After 2 h of bacteria-cell contact, coverslips were washed with PBS to remove non-associated bacteria, and cells fixed with methanol/acetone (80:20) for 20 min at -20°C, washed and blocked for 10 min. Coverslips were incubated 1 h with rabbit anti-S. suis serum and with a monoclonal antibody against swine MHC class II antibody . After washing, coverslips were incubated with the secondary antibodies Alex-Fluor 488 goat anti-rabbit IgG (S. suis) and Alex-Fluor 568 goat anti-mouse IgG (DC MHC-II) for 30 min, washed and mounted on glass slides with moviol containing DABCO and DAPI to stain the nuclei. Secondary antibodies were from Invitrogen, CA, USA. The polyclonal antiserum against S. suis serotype 2 recognizes both wild type and non encapsulated mutants at similar levels and has previously been used in other studies with the same strains [For confocal microscopy analysis, cells were placed on coverslips and infected with the  strains ,22.S. suis strains were incubated with DCs for 4 h or 2 h, respectively. After two washes with PBS, the samples were fixed for 1 h at room temperature with 2% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) and were then postfixed for 45 min at room temperature with 2% osmium tetroxide. Samples were then postfixed in 2% (vol/vol) osmium tetroxide in deionized water. Specimens for TEM were dehydrated in a graded series of ethanol solutions and embedded with LR White resin. Thin sections were cut with a diamond knife and were poststained with uranyl acetate and lead citrate. Samples were observed with an electron microscope model JEOL JEM-1230. Samples for SEM were dehydrated in a graded series of ethanol solutions and covered with gold after critical point drying and were examined with a Hitachi S-3000 N microscope.For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), S. suis (MOI: 0.001). Supernatants were collected at 16 h after infection to measure cytokines by ELISA and cells were harvested for analysis of co-stimulatory molecules by FACS. Lactate dehydrogenase (LDH) release measurement assay was used to measure cytotoxicity levels  as previously described [Escherichia coli 0111:B4 lipopolysaccharide (LPS) at 1 μg/mL  was used as positive control.DCs were resuspended and stimulated with Levels of IL-1β, IL-6, IL-8, IL-12p40 and TNF-α in cell culture supernatants were measured by sandwich ELISA using pair-matched antibodies from R&D Systems , according to the manufacturer's recommendations. Twofold dilutions of recombinant porcine cytokines were used to generate the standard curves. Sample dilutions giving optical density readings in the linear portion of the appropriate standard curve were used to quantify the levels of each cytokine. The results are from at least three independent experiments with at least two technical replicates.+/MHC-I+/MHC-II+/CD1c+/CD14+/CD16+/CD163low/CD4-/Cd11R1- cells, as previously described [DCs were phenotypically characterized for the following markers: SWC3, MHC-I, MHC-II, CD1c, CD4, CD11R1, CD14, CD16, CD80/86 and CD163, and were shown to be composed of SWC3escribed ,19. Supeescribed ,23,24, a5 cells were incubated with the appropriate antibody for 1 h on ice followed by washing and staining for 1 h on ice with the secondary antibody goat anti-mouse IgG-PE . After washing, cells were resuspended in sorting buffer for FACS analysis. Flow cytometry was performed using a FACSCalibur instrument . A total of 20 000 gated events were acquired per sample and data analysis was performed using CellQuest software. Quadrants were drawn based on FITC- and PE-control stains and were plotted on logarithmic scales. The results are from at least three independent experiments.For cell surface staining, 2.5 × 10S. suis strains 31533 and B218 (MOI: 0.001) for 2 h, 4 h, 10 h and 16 h. Cells stimulated with specific ligands of the TLR family were used as controls. PAM3CSK4 , FSL-1  and ultra pure LPS  were used and were obtained from InvivoGen . Following infection, medium was removed and cells were washed. Total cellular RNA was prepared from cells using Trizol reagent  according to the manufacturer's instructions. Next, 1 μg of total RNA was reverse-transcribed with the QuantiTect reverse transcription kit . The cDNA was amplified using the SsoFast™ EvaGreen® Supermix kit . The PCR amplification program for all cDNA consisted of an enzyme activation step of 3 min at 98°C, followed by 40 cycles of a denaturing step for 2 s at 98°C and an annealing/extension step for 5 s at 58°C. The primers used for amplification of the different target cDNA are listed in Table Ct -ΔΔmethod. Peptidylprolyl isomerase A (PPIA) was used as the normalizing gene to compensate for potential differences in cDNA amounts. The non-infected DC group was used as the calibrator reference in the analysis. The results are from at least three independent experiments.DCs were infected with t-test. Data from RT-PCR were subjected to ANOVA procedures. A p value < 0.05 was used as threshold for significance. All experiments were repeated at least three times.All data are expressed as mean ± SEM. Data from the phagocytosis assay and ELISA tests were analyzed for significance using the Student's unpaired S. suis, pre-opsonized or non-opsonized bacteria were incubated with DCs for different time periods. As shown in Figure S. suis by swine DCs. Serum components did not seem to influence S. suis phagocytosis levels by DCs, as no significant differences were noticeable between pre-opsonized and non-opsonized bacteria for either the wild-type strain or the non-encapsulated mutant .To analyze the intracellular fate of bacteria once internalized, we modified the phagocytosis assay in order to quantify bacterial intracellular survival over time. Precisely, after 60 min incubation of S. suis-infected DCs were measured at 16 h after stimulation. Time and bacterial dose for the cytokine stimulation assays were chosen using the absence of cytotoxicity and significant activation as the selection criteria (data not shown). Our results show that DCs produced significant amounts of these cytokines after exposure to S. suis wild-type strain compared to control, non-activated cells. However, the non-encapsulated strain induced significantly higher levels of all cytokines tested, except for IL-1β, compared to the wild-type strain CSK, FSL-1 and LPS were used as positive controls for TLR2/TLR1, TLR2/TLR6 and TLR4, respectively. As shown in Figure S. suis wild-type strain induced significant up-regulation of TLR2 and TLR6 mRNA by DCs at 16 h and 10 h of infection, respectively. Similarly, the non-encapsulated strain activated both TLR2 and TLR6 within 10 h of infection . Finally, the expression of TLR4 was not up-regulated in the presence of S. suis even though an upregulation was noticeable with the positive control LPS (data not shown).To analyze whether n Figure . As low S. suis to induce surface expression of the co-stimulatory molecules MHC-II and CD80/86 by DCs was investigated by FACS 16 h after stimulation  were described [S. suis uses an arsenal of different virulence factors to modulate mDC functions and escape immune surveillance, mainly by modulating cytokine release and escaping opsono-phagocytosis. However, it is important to confirm S. suis modulation of DC activation in the natural host, the swine. The present work demonstrates for the first time that S. suis interacts with porcine DCs and modulates their maturation and activation.n health ,3,4. Hownfection ,26, and escribed . The resS. suis and to evaluate the role of the capsular polysaccharide in this process. We observed that the presence of the capsular polysaccharide protects S. suis from DC phagocytosis, both under opsonic and non-opsonic conditions. This confirms the role of the capsular polysaccharide as an anti-phagocytic factor. In agreement, previous studies (using the same wild type and mutant strains included in this study) with monocytes/macrophages, neutrophils and mDCs demonstrated that the capsular polysaccharide reduces S. suis phagocytosis [S. suis in vivo. Indeed, the non-encapsulated mutant strain used in this study was shown to be avirulent and rapidly eliminated from the bloodstream in a porcine model of infection [S. suis phagocytosis by DCs, the intracellular survival assay showed that once internalized, both encapsulated and non-encapsulated strains are equally destroyed. Our previous data with mDCs showed that both encapsulated and non-encapsulated S. suis localizes within LAMP+ vacuoles suggesting phagosome fusion with lysosomes leading to bacterial destruction [S. suis extracellular localization confers to this pathogen a survival advantage and the capsular polysaccharide is essential to it. Wild-type S. suis was shown to trigger DC release of IL-1β, IL-6, IL-8, IL-12p40 and TNF-α. These cytokines, among others, were recently shown to be released by mDCs following stimulation with wild-type S. suis strain 31533 [S. suis cell wall mutant strains [S. suis cell wall components, studies to date have identified two cytokines for which the capsular polysaccharide is required for optimal induction, MCP-1 and IL-1β [S. suis capsular polysaccharide was also shown not to interfere with the production of IL-1β production by swine DCs. The molecular pathways underlying the capsular polysaccharide contribution to IL-1β and MCP-1 release are under evaluation. Finally, and accordingly to data from Devriendt et al., porcine DCs were low responsive to LPS [We used a phagocytosis assay, combined with confocal and electron microscopy to measure the ability of DCs to internalize ocytosis ,7,21,27.nfection . Despitetruction ,30. Altoin 31533 ,31,32. TS. suis [S. suis. It should be noted, however, that interactions between S. suis and epithelial cells can highly differ from those observed with DCs. It has also been demonstrated that stimulation of human monocytes by whole encapsulated S. suis or its purified cell wall components influences the relative expression of TLR2 mRNA [S. suis. Since this response was completely inhibited in MyD88-deficient macrophages, other TLRs could be involved in cytokine production induced by S. suis. In addition, it was demonstrated that the presence of the capsular polysaccharide modulates interactions between S. suis and TLRs, as uncovered cell wall components were shown to induce cytokine production through TLR2-dependent and -independent pathways [S. suis invasion of the central nervous system, transcriptional activation of TLR2, TLR3 and CD14 has been observed in a mouse model of infection [S. suis stimulation of cells of porcine origin.In addition to cytokine production, both the wild-type strain and its non-encapsulated mutant strain increased the expression of TLR2 and TLR6 mRNA. No differences were noticeable between the two strains for the expression of TLR2. However, the expression of TLR6 was increased more rapidly after DC infection with the non-encapsulated strain than that observed for the wild-type strain. This activation pattern is in agreement with that recently reported by Wichgers Schreur et al. , who shoS. suis . HoweverLR2 mRNA . MoreoveLR2 mRNA . Mouse mpathways . Finallynfection . This stS. suis to induce the maturation of DCs was also investigated by evaluating the surface expression of the co-stimulatory molecules CD80/86 and MHC-II on swine DCs. S. suis wild-type strain failed to induce the expression of either CD80/86 or MHC-II on DCs. The capsular polysaccharide was shown to be responsible for the impaired expression of CD80/86 on DCs and also seems to interfere, at least in part, with MHC-II expression. This differed with results obtained with mDCs where wild-type S. suis induced mDC maturation levels similar to those observed with the non-encapsulated mutant [S. suis-induced MHC-II expression by DCs derived from different pigs. This could be related to the fact that genes of the MHC complex have high levels of polymorphism [S. suis would successfully colonize only some piglets and not others, and why some animals will only be healthy carriers and will never develop disease whereas others will develop bacteremia, sometimes septicemia and finally meningitis [S. suis pathogenesis.The ability of d mutant . These dmorphism ,36. Highmorphism -40. The morphism . The higningitis . As indiS. suis interacts with swine origin DCs and suggest that these cells might play a role in the development of host innate and adaptive immunity during an infection with S. suis serotype 2. S. suis resists phagocytosis but is able to activate the release of pro-inflammatory cytokines by DC mainly through the activation of TLRs 2 and 6. In fact, S. suis capsular polysaccharide was shown to modulate most interactions with DCs by protecting bacteria against phagocytosis, reducing the level of cytokine production and preventing the surface expression of co-stimulatory molecules. Overall, capsular polysaccharide-impaired S. suis interactions with DCs would result in low bacterial up-take as well as low DC activation and maturation which might translate in reduced antigen processing and T cell activation, although this should be confirmed. The capsular polysaccharide could therefore be considered as an escape mechanism for S. suis. It is important to note that since none of the non-encapsulated mutants available in the literature (including the one used in this study) could so far be successfully complemented (showing restoration of capsule production), a certain additional role of other unknown mutation in those mutants cannot be completely ruled out. The importance of DCs on the efficacy of the immune system has been clearly demonstrated in the last years [To conclude, our results show for the first time that st years ,43. HoweThe authors declare that they have no competing interests.MPL participated in the design and coordination of the study, carried out the study, performed the statistical analysis, and wrote the manuscript. CL participated in the design of the primers and experiments for RT-PCR. CS participated in the collect of swine femurs. TM and MM participated in the generation of bmDCs from swine femurs. MS and MG conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.